This invention relates to a method of linking primary aromatic amines to carrier proteins by photochemical reactions in order to produce antibodies against the amine haptens.
More specifically, the invention relates to a method for coupling aromatic nitro- or amine-containing drugs or other compounds to carrier proteins for the purpose of raising antibodies against the coupled compound. The conjugated proteins can then be used to produce antibodies specific for the coupled haptenic group or, if the drugs are coupled to antibodies, these may then be used to increase the efficacy of drug delivery to targeted sites and decrease toxicity. The coupling reaction is rapid and occurs under mild conditions which do not result in denaturation or loss of function of the carrier proteins. The coupling procedure provides for high labeling densities which can be obtained without loss of protein function.
Two methods are primarily used to conjugate primary aromatic amines to carrier proteins. They are the diazocoupling method (Inman, et al., 1973, Immunochem. 10, 153-163) and the isocyanate method (Spragg, et al., 1966, J. Immunol. 96, 865-871). However, there are some disadvantages to the use of these methods. The conjugated proteins prepared by these methods may sometimes fail to elicit an anti-hapten antibody response, which may be attributed to the highly alkaline conditions required for coupling. It is also difficult to prevent side reactions associated with diazonium coupling, which may result in extensive precipitation. Furthermore, the bonds formed by the diazonium salts are easily cleaved. Finally, the extent of the diazocoupling is mainly dependent on the presence of tyrosine and histidine residues in the carrier protein, which further restricts the applicability of this approach. The isocyanate method of Creech, H. J. (1952) Cancer Res., 12, 557-564 has been used for conjugating carcinogenic aromatic primary amines to carrier proteins. This method involves the derivatization of the primary aromatic amine to form the isocyanate followed by coupling to the .SIGMA.-amino group of a lysine residue. Fraenkel-Conrat, H. L. (1944) J. Biol. Chem. 152, 385-389 reported that isocyanates react with the --SH groups of cysteine and Miller, et al. (1941) J. Biol. Chem. 141, 905-920 have reported that isocyanates also react with --OH groups of tyrosine. However, Creech, et al. (1941) J. Am. Chem. Soc. 63, 1670-1673 were unable to conjugate zein with isocyanates even under favorable experimental conditions. Additionally, attempts by Creech et al. (1941) J. Am. Chem. Soc. 63, 1661-1669 to increase the epitope density (the number of haptenic groups attached per molecule carrier) beyond a certain level resulted in denaturation of the protein. The coupling conditions for this method, stirring the hapten and carrier protein at alkaline pH and 4.degree. C. overnight, are similar to those for the diazocoupling method and present the same problems. The specific functional group requirements of the isocyanate coupling procedure also restrict the applicability of this method.
Photolabeling techniques have previously not been used to conjugate primary aromatic amines with carrier proteins in order to elicit antibodies against the hapten. Primary aromatic amines may be easily derivatized to azido compounds which are reasonably stable in the dark and at low temperatures. The resultant azido derivatives are photolabile when irradiated with ultraviolet (UV) light in ethanolic or aqueous solutions, giving rise to highly reactive nitrene radicals which can undergo insertion reactions.
There is a need to conjugate aromatic nitro- or amine-containing compounds to carrier proteins in a relatively short period of time at physiological pH.